Male gametogenesis in maize has been well characterized biochemically and cytologically, but our understanding of the molecular events controlling this key process in the angiosperm life cycle is at present minimal. The complex differentiation of a maize pollen mother cell into the highly specialized trinucleate pollen grain suggests that male gametogenesis involves the sequential production of many gene products. However, only a few genes involved in male gametogenesis in flowering plants have been isolated.
Evidence for the sequential expression of genes in the anther comes from the identification of two classes of transcripts expressed during male gametogenesis (Mascarenhas 1990); the "early" genes are expressed post-meiotically with their levels increasing before declining as the pollen grains mature. The "late" genes are switched on after microspore mitosis and increase to a maximum in mature pollen. It has been estimated (Willing et al. 1988) that around 20,000 genes are expressed in maize pollen and possibly up to 10% are pollen-specific (Stinson et al. 1987). The only reported pollen-specific genes cloned from maize are Zm13 (Hanson et al. 1989) and Zm58 (Mascarenhas 1990). The expression of these genes can first be detected following microspore mitosis and they increase to a maximum in mature pollen. Zmc13 shows homology in its predicted amino acid sequence to Kunitz trypsin inhibitor proteins and also to a tomato anther-expressed gene LAT52 (Hanson et al. 1989). Zm58 shows homology in its predicted amino acid sequence to pectate lyases (Mascarenhas 1990). It is suspected that the products of genes in this class have a role in pollen germination and/or pollen tube growth, whereas products of the "early" genes are more likely to be involved in microspore development.
Sequence comparisons and mutagenesis experiments have implicated certain DNA sequence motifs in the control of pollen expression of the LAT genes in tomato (Twell et al. 1991). Sequence comparisons alone have been used for the putative identification of a region involved in anther specific expression in petunia (van Tunen et al. 1989).
At present, little is known of possible cis-acting sequences in the maize genome that may be responsible for the tissue-specific expression of functions peculiar to pollen development. However, the maize clone, Zm13, does possess several regions of homology with the proposed pollen boxes identified in the tomato LAT genes.
Rogers et al. 1991 have described the isolation of a cDNA clone, 3C12 (incomplete at its 5' end), from maize which shows homology in its predicted amino acid sequence to polygalacturonases (PG) from both eucaryotic and procaryotic species, including a pollen-expressed PG from Oenothera organensis (Brown and Crouch 1990). Polygalacturonase has been detected in the pollen of maize and other monocotyledonous plant species (Pressey and Reger 1989). It is possible that the function of polygalacturonase in the pollen grain is in the growth of the pollen tube down the silk by hydrolyzing pectin and providing components which can be used as precursors of the pollen tube cell wall, or it may be involved in the degradation of cellular material within the silk to allow penetration of the tube. Allen and Lonsdale (submitted for publication) have also described the isolation, by use of the 5' end of the maize clone 3C12 as a probe, of 4 genomic PG clones. Analysis of the sequence of three of the PG genomic clones suggests that polygalacturonases are members of a multigene family (Allen and Lonsdale submitted for publication). The polygalacturonase gene isolated from tomato and involved in fruit-ripening (Grierson et al. 1986) is present in a single copy in the tomato genome. Brown and Crouch (1990) isolated at least six unique cDNA clones with homology to polygalacturonase from a pollen cDNA library of Oenothera organensis, suggesting that multiple PG genes are expressed in pollen of that species.
Of the 2,000 possible pollen-specific genes of maize, only two have been characterized. U.S. Pat. No. 5,086,169 ('169) discloses the nucleotide sequence of an isolated pollen-specific promoter called Zm13 from an unidentified gene expressed in corn. The pollen-specific promoter sequence consists of 1315 base pairs upstream from a region of DNA which hybridizes to mRNA found only in pollen. This is the same pollen-specific promoter described by Hanson et al. (1989).
It is an object of the present invention to isolate and characterize a DNA sequence which is capable of acting as a pollen-specific promoter. More particularly, it is an object of the invention to isolate and characterize a pollen-specific promoter region taken from a pollen-specific polygalacturonase gene.